Discussion
The concept that the distribution of WHY1 between the nuclei and chloroplasts plays a key role in the regulation of plant development has arisen in recent years (Ren et al., 2017; Lin e al., 2019). The phosphorylation of the WHY1 protein favours partitioning to nuclei, a process that increase with leaf age (Guan et al ., 2018). However, little information is available on the distribution of WHY1 between the nuclei and proplastids of developing leaves. The immunogold labelling analyses reported here revealed that over 60% of the WHY1 protein in the basal sections of the leaves was located in the nuclei (Fig. 2). Hence, it is likely that WHY1 fulfils its functions in the nuclei of developing leaves as well as the plastids. Chloroplast development is delayed in the absence of WHY1, a process that has largely been ascribed to the functions of WHY1 in chloroplasts (Prikryl, Watkins, Friso, van Wijk, & Barkam, 2008; Kurpinska et al., 2019). However, WHY1 functions as a transcriptional activator in the nucleus, binding to the AT-rich region of the kinesin gene promoter to activate kinesin gene expression (Xiong et al., 2009), and to the GTCAAT motif of the S40  promoter (Krupinska et al., 2014), and to a combination motif of GTNNNAAT and AT-rich motif of downstream target genes, such as WRKY53WRKY33SPO11 , and PR1  to regulate leaf senescence and other processes inA. thaliana(Miaoet al.,2013; Renet al., 2017; Huang et al. , 2018). Our transcriptome profiles of the three developmental regions in the W1-1 and W1-7 leaves compared to the wild type leaves provide some insights into how the developmental pattern of transcripts is changed in the absence of WHY1. While most transcripts exhibited similar patterns of abundance in the developmental profiles of all genotypes there were some clear exceptions such as a GNC transcription factor regulating stomatal development, greening and chloroplast development and NAC1, an auxin-regulated senescence-associated transcription factor, two transcripts encoding ARF transcriptions factors with functions in leaf morphogenesis and development and two transcripts with similarity to Arabidopsis GLK1 that encodes a transcription factor required for the expression of nuclear encoded photosynthetic genes (Waters et al., 2009). Moreover, SAG12 transcripts were more abundant in all the sections of the W1-7 line than the other genotypes. These differences are indicative of divergent chloroplast development programmes in leaves deficient in the WHY1 protein.
The WHY1 protein binds to ERF-binding cis elements in the promoter regions of genes such as ERF109 (REDOX RESPONSIVE TRANSCRIPTION FACTOR 1, RRTF1) (Miao et al., 2013. ERF109 is involved in plant stress responses and participates in reactive oxygen species (ROS) signalling and the regulation of developmental programs, such as jasmonate-dependent initiation of lateral root development (Huanget al. , 2019). WHY proteins have previously been reported to be involved in the regulation of shoot and root development. For example, WHY2 was shown to be a major regulator of root apical meristem development (McCoy et al., 2021). Similarly, the expression of WHY1 (nWHY1) in the nucleus of Arabidopsis why1 mutants led to changes in the levels of transcripts associated with plant development during early growth, whereas expression of WHY1 in plastids increased the abundance of transcripts associated with salicylic acid synthesis (Lin et al., 2020). The binding of WHY proteins to the PB element of the9-cis epoxycarotenoid dioxygenase (NCED)1 gene in cassava activated expression leading to increased abscisic acid levels (Yan,et al., 2020). Hence, the presence of WHY proteins in the nucleus clearly influences the expression of genes involved in the synthesis of phytohormones that control plant growth and defence. The primary cause of the delay in greening observed in the barley leaves lacking WHY1 may therefore result from the absence of WHY1-dependent regulation of nuclear gene expression.
The action WHY1 as a transcription factor in the nucleus also regulates the expression of genes associated with photosynthesis and carbon metabolism. For example, WHY1 binds to the promoter of the rbcS gene that encodes the small subunit of the potato ribulose-1, 5-carboxylase, oxygenase under cold stress (Zhuang et al. , 2020), while WHY2 binds to the promoters of the SWEET11/15 genes that encode sucrose transporters, leading to the modulation of starch allocation and silique development (Huang et al ., 2020). Here we report that the absence of WHY1 has a significant impact on the levels of transcripts encoding enzymes associated with the Calvin cycle, starch and sugar metabolism, glycolysis, the TCA cycle and amino acid metabolism, many of which were more abundant in WHY1-deficient leaves than the wild type. However, transcripts encoding enzymes such as β-amylase were less abundant in WHY1 knockdown lines. WHY1 is known to bind to the ERE-like element of the AMY3-L promoter, activating the expression of amylase and starch degradation. WHY1 also binds to the ERE element of the ISA2 promoter to inhibit isoamylase-mediated starch-synthesis (Zhuang et al. , 2019). The absence of WHY1 from the nuclei of developing barley leaves could therefore lead to the observed changes in primary metabolites reported here. For example, all the metabolites of the TCA cycle that were detected were significantly lower in WHY1 knockdown leaves than the wild type, as were GABA. Other amino acids such as glycine, valine, leucine, threonine and isoleucine were higher in WHY1-deficient leaves than the wild type. It may be that WHY1 can bind to promotors of a wide range of housekeeping genes in the nucleus, to modulate their expression in response to developmental and environmental signals.
The WHY1-deficient barley leaves showed delayed chloroplast ribosome formation and acquisition of photosynthetic activity suggesting that WHY1 contributes to the coordination of nuclear and plastome gene expression (Krupinska et al. , 2019). The role of the WHY proteins in organelle to the nucleus retrograde signalling has long been suspected (Foyer et al., 2014) but remains to be established. Similarly, the factors that determine the partitioning of WHY1 between the chloroplasts and nuclei remain to be fully characterised. The compartmentation of WHY1 between the plastids and nuclei is influenced by protein phosphorylation, particularly as the leaves enter senescence (Renet al ., 2017). Phosphorylation of the WHY1 protein by CIPK14 kinase or oxidation by the addition of hydrogen peroxide causes a re-distribution of WHY1 from the plastids to the nucleus (Ren et al., 2017; Lin et al., 2019). However, little information is available about the regulation of WHY1 partitioning in other situations such as the bases of developing leaves. Earlier evidence indicated that WHY1 can move from the plastids to the nucleus (Isemer et al., 2012). Direct transfer of WHY1 from the plastids to the nuclei through contact sites or stromules (Hanson et al, 2018) is possible but remains to be proven. Moreover, the plastid-localized WHY1 affects miRNA biogenesis in the nucleus (Swida-Barteczka et al., 2018) suggesting that WHY1 influences chloroplast to nucleus signalling.
In summary, our understanding of WHY protein functions have greatly increased in recent years, as has our knowledge of the flexibility of their localisation and overlap of functions. The data presented here provides new insights into the intracellular localization of the WHY1 in developing leaves, highlighting how WHY1 in the nucleus might control chloroplast development.